Improved embryogenesis process for initiation and maturation

ABSTRACT

A method of producing mature somatic embryos (especially of conifers) to provide unexpected yield advantages comprising the steps of: (1) placing explants of the immature embryos on and/or in an initiation medium (the medium itself being part of the invention) or on a nurse culture itself on or in the initiation medium, (2) allowing the initiation to take place, and (3) (whether after optional storage and maintenance or not) maturing the initiated embryos on an appropriate maturation medium, wherein: (a) the initation and maturation medium may be the same or different, and wherein (b) (i) at least the initiating medium contains ABA and at least one amino acid, or (b) (ii) at least the initiating medium contains at least one amino acid and no ABA, and (c) at least the maturation medium contains ABA and at least one amino acid.

[0001] The present invention relates to an improved conifer or otherwoody species embryogenesis process for initiation and maturation.

[0002] The initiation and maturation of embryogenic tissue and somaticembryos respectively has hitherto been part of a stagewise process fromwhich explant to embryogenic tissue through to germination of somaticembryos and growth of somatic seedlings in the field has involved the 8stages referred to hereinafter.

[0003] 1. Initiation of embryogenic tissue

[0004] 2. Maintenance of embryogenic tissue

[0005] 3. Development of embryogenic tissue

[0006] 4. Maturation of somatic embryos

[0007] 5. Starvation and storage of somatic embryos (see New ZealandPatent Specification No. 272210)

[0008] 6. Germination of somatic embryos

[0009] 7. Growth of somatic seedling in greenhouse

[0010] 8. Growth of somatic seedlings in field

[0011] Protocols for somatic embryogenesis for conifers typicallyinvolve several stages from initiation of embryogenic tissue through tosomatic embryo maturation and germination. Patents providing backgroundas to the use of embryogenesis to create somatic embryos includeWO95/14373, U.S. Pat. No. 5,036,007, U.S. Pat. No. 5,034,326, U.S. Pat.No. 5,041,382, U.S. Pat. No. 4,957,866, AU 37150/93, U.S. Pat. No.5,294,549, South Africa 93/4807, and USA (CIP) 08/219879 (unpublished).

[0012] For initiation of conifer embryogenic cell lines wholegametophytes containing immature fertilised embryos or dissectedimmature fertilised embryos are used as explants. Explants are placed onseveral different “initiation media” to initiate embryogenic tissueeither with or without growth regulators. The average percentageinitiation over an entire seasonal initiation for radiata pine usingwhole gametophytes as explants has varied from 5-10%. The bestpercentage initiation has vaned from 6-34% for the best developmentalwindow of sampling the explants from each seedlot. An initiated cellline is defined as an established and maintained cell line.

[0013] For maturation of conifer somatic embryos, initiated embryogeniccell lines are placed onto several media to maintain and develop theembryogenic tissue and multiply the number of embryo initials.Embryogenic tissue is then generally placed on a “maturation medium” toencourage the tissue to form mature embryos. Typically this maturationmedia contains abscisic acid (ABA). The percentage of initiated celllines that are able to continue growth and form mature somatic embryosis typically 1-10%. Higher percentages of up to 25% have been obtainedwhen a selection of 20-25% of the maintained embryogenic cell lines areplaced on maturation media, ie; not all cell lines initiated aresubsequently suitable for placement on maturation media.

[0014] The percentage of cell lines that are currently initiated usingpreviously patented or published protocols is too low for adequateclonal representation within a family or cross and genetic diversity isnot maintained satisfactorily.

[0015] Furthermore, from the embryogenic cell lines initiated therepresentation of clones forming mature embryos within a family isfurther reduced.

[0016] It is desirable from the perspectives of clonal testing, geneticdiversity, process efficiency and cost effectiveness to have at least50% initiation of clonal embryogenic cell lines and at least 30%formation of mature somatic embryos from those initiated embryogeniccell lines.

[0017] The present invention can achieve or at least approach this.

[0018] The present invention relates to various procedures and relatedmethods and includes an embryogenic initiation medium which will resultin changes to at least stages 1, the prospect of a merging of steps 2and 3 with each of stages 1 and 4 and additionally potentiates theoutcome at maturation step 4 for that embryogenic tissue capable ofgenerating somatic embryos, the present invention therefore providing anincreased efficiency over the prior art procedures.

[0019] It is to this that the present invention is directed.

[0020] In a first aspect the present invention consists in a method ofinitiating embryogenic tissue from a source of immature embryos of aconifer or other woody species, said method comprising:

[0021] placing explants of the immature embryos on and/or in aninitiation medium or on a nurse culture itself on or in the initiatingmedium, and

[0022] allowing sufficient time for initiation to take place,

[0023] wherein (i) the initiation medium contains ABA and/or at leastone amino acid, or (ii) the initiation medium containing just aminoacids.

[0024] As used herein “woody species” includes the groups of speciesEucalyptus family, Proteaceae, Myrtaceae, Rosaeceae, Punicaceae, etc.

[0025] Preferably said conifer immature zygotic embryo explants for theinvention include those of Pinus radiata (or Monterey pine), hybrids ofPinus radiata and genetically modified Pinus radiata. This procedure isalso applicable to other conifer species, viz, loblolly pine, Douglasfir, spruce species etc. and, of course, hybrids or genetically modifiedversions thereof.

[0026] Reference to “on” and “in” in respect of the media at leastcontemplates the use of gelled and/or liquid media.

[0027] Preferably said explant is not the whole megagametophyte andpreferably is the dissected fertilised embryos at the bullet stage andbefore the pre-cotyledonary stage, 500- (if radiata pine) celled embryohead developmental stage and most probably different called embryo headcounts for other species which have different sized and shaped embryos.

[0028] Preferably the initiation medium does not containtraditional/conventional plant growth regulators such as auxins andcytokinins (eg; 2, 4-D, IAA, NAA, IBA, BAP, 2-IP, Zeatin, TDZ, etc). Norpreferably is it a medium for initiation with no growth regulators asoutlined in South Africa 93/4807. But it does contain ABA and/or one ormore amino acids.

[0029] Preferably ABA (Abscisic Acid) is present.

[0030] Preferably where said initiating medium is also to be used as thematuration medium ABA is present.

[0031] In another preferred form Abscisic Acid (ABA) may be absent butat least one amino acid is present. Preferably said amino acid that ispresent is one or more of the amino acids Arginine, Asparagine,Glutaine, Citrulline, Ornithine, Lysine, Alanine and Proline.

[0032] Preferably Glutamine is present.

[0033] Preferably at least one of Asparagine and Arginine is alsopresent.

[0034] Preferably Glutamine, Asparagine and Arginine are present.

[0035] Preferably the initiation medium contains both ABA and at leastone of the aforementioned amino acids and optionally several or all ofthe aforementioned amino acids.

[0036] Preferably said initiation medium includes in addition to saidABA and/or said at least one amino acid and other nutrient sources suchas, for example, a source of essential macro and micro elements,vitamins, carbohydrates, inositol etc.

[0037] Preferably the initiation medium includes inorganic ions in thefollowing ranges in concentration of the more significant ions in apreferred medium CONCENTRATION RANGE ION (mmoles/l) NO₃ 4.27 NH₄ 0.5-6.8Ca   0-0.9 Fe   0-0.15 Na 0-7 Zn    0-0.135 Cu   0-0.05 Mg   0-3.24

[0038] Preferably said ion concentrations are CONCENTRATION RANGE ION(mmoles/1) NO₃ about 17.8 NH₄ about 1.96 Ca about 0.17 Fe about 0.10 Naabout 3.85 Zn about 0.09 Cu about 9.61 × 10⁻³ Mg about 1.62

[0039] In another embodiment preferably also present in the medium arethe following inorganic ions or the total presence of inorganic ions isas follows ION CONCENTRATION (mmoles/l) NO₃ 17.80 NH₄ 1.96 TOTAL 19.76 NP 1.96 K 14.16 Ca 0.17 Mg 1.62 Cl 3.42 × 10⁻¹ Fe 0.10 S 1.83 Na 3.85 B0.13 Mn 1.62 × 10⁻² Zn 0.09 Cu 9.61 × 10⁻³ Mo 8.27 × 10⁻⁴ Co 8.41 × 10⁻⁴I 6.02 × 10⁻³

[0040] Preferably also included are 5 g/l-50 g/l (w/v) Sucrose(preferably about 30 g/l).

[0041] Preferably it also includes 3-8 grams gellan gum per litre(preferably about 5 grams) or other gelling agent (eg; agar or other).

[0042] Preferably it also includes 5 mg/l-50 mg/l (w/v) Abscisic acid(ABA) (preferably about 15 mg/l).

[0043] Preferably the amino acids are present in the following rangesION CONCENTRATION RANGE (mg/l) Arginine  500-2,000 Asparagine1,000-4,000  Glutamine 1,000-10,000 Citrulline 0-50 Ornithine 0-50Lysine 0-50 Alanine 0-50 Proline 0-50

[0044] Preferably arginine is about 700, preferably asparagine is about2,100 and preferably glutamine is about 7,300.

[0045] In another aspect the present invention consists in a method ofinitiation of embryogenic tissue from a source of immature conifer orother woody species, said method comprising:

[0046] placing explants of the immature fertilised embryos (directly orindirectly eg; nurse culture) on and/or in an initiation medium, and

[0047] allowing sufficient time for the initiation to take place,

[0048] wherein the initiation medium contains ABA and amino acids, or

[0049] wherein the initiation medium contains amino acids and no ABA.

[0050] Preferably conifers are the source of the explants.

[0051] Preferably the sufficient time is of the order of about 4-6weeks.

[0052] Preferably the environment is in a sterile tissue culture vesselin a controlled environment at 15-28° C.

[0053] In a further aspect the present invention consists in a method ofinitiation of embryogenic tissue from a source of immature conifer orother woody species embryos, said method comprising

[0054] placing explants of the immature fertilised embryos (directly orindirectly eg; nurse cells) on and or in an initiation medium, and

[0055] allowing sufficient time for the initiation to take place,

[0056] wherein the initiation medium is; Final Rate per Litre (mg)Potassium Nitrate (KNO₃) 1431 Magnesium Sulphate (MgSO₄.7H₂O) 400 SodiumNitrate (NaNO₃) 310 Ammonium Dihydrogen Phosphate (NH₄H₂PO₄) 225 CalciumChloride (CaCl₂.2H₂O) 25 Zinc Sulphate (ZnSO₄.7H₂O) 25 Boric Acid(H₃BO₃) 8.0 Manganese Sulphate (MnSO₄.H₂O) 2.72 Copper Sulphate(CuSO₄.5H₂O) 2.4 Potassium Iodide (KI) 1.0 Cobalt Chloride (CoCl₂.6H₂O)0.2 Molybdic Acid (Na₂MoO₄.2H₂O) 0.2 EDTA - Disodium Salt 40 IronSulphate 7H₂O 30 Nicotinic Acid 5.0 Thiamine HCl 5.0 Pyridoxine 0.5Inositol 1000 Arginine 700 Asparagine 2100 Glutamine 7300 Citrulline3.95 Ornithine 3.80 Lysine 2.75 Alanine 2.0 Proline 1.75 Abscisic Acid 5to 50 Sucrose 5,000 to 50,000 Gelling Agent (GELRITE ™) 3,000 to 8,000

[0057] Preferably the sucrose is about 30,000, the gelling agent isGELRITE™ about 4,500, and the abscisic acid is about 15.

[0058] In this respect reader is referred to South African PatentSpecification No. SA 93/4807 where media is used for maturation only.The present invention recognises the usefulness and advantages of amedia (not necessarily including ABA) for initiation and also (whenpreferably including ABA) for maturation.

[0059] In a further aspect the invention is a method of producing maturesomatic embryos comprising the steps

[0060] (1) placing explants of the immature embryos on and/or in aninitiation medium or on a nurse culture itself on or in the initiationmedium,

[0061] (2) allowing the initiation to take place,

[0062] (3) (whether after optional storage and maintenance or not)maturing the initiated embryos on an appropriate maturation medium, andwherein

[0063] (a) the initiation and maturation medium may be the same ordifferent, and wherein

[0064] (b) (i) at least the initiating medium contains ABA and at leastone amino acid, or

[0065] (b) (ii) at least the initiating medium contains at least oneamino acid and no ABA.

[0066] Preferably said at least one amino acid is selected from thegroup Arginine, Asparagine, Glutamine, Citrulline, Ornithine, Lysine,Alanine and Proline.

[0067] Preferably the maturation medium contains ABA and at least oneamino acid.

[0068] The invention also consists in embryos thus matured.

[0069] In still a further aspect the present invention consists in aninitiation and/or maturation media for embryogenic tissue, said mediumcomprising in addition to a presence of ABA and at least one amino acid,inorganic ions in the following ranges in concentration of the moresignificant ions in a preferred medium. ION CONCENTRATION RANGE(mmoles/l) NO₃  4-27 NH₄ 0.5-6.8 Ca 0.01-0.9  Fe 0.025-0.15  Na 0.5-7  Zn 0.023-0.135 Cu 6 × 10⁻⁴-5 × 10⁻² Mg 0.405-3.24 

[0070] Preferably said ion concentrations are ION CONCENTRATION RANGE(mmoles/l) NO₃ about 17.8 NH₄ about 1.96 Ca about 0.17 Fe about 0.10 Naabout 3.85 Zn about 0.09 Cu about 9.61 × 10⁻³ Mg about 1.62

[0071] Preferably said ion concentrations are as follows IONCONCENTRATION (mmoles/l) NO₃ 17.80 NH₄ 1.96 TOTAL 19.76 N P 1.96 K 14.16Ca 0.17 Mg 1.62 Cl 3.42 × 10⁻¹ Fe 0.10 S 1.83 Na 3.85 B 0.13 Mn 1.62 ×10⁻² Zn 0.09 Cu 9.61 × 10⁻³ Mo 8.27 × 10⁻⁴ Co 8.41 × 10⁻⁴ I 6.02 × 10⁻³

[0072] Preferably 5 g/l-50 g/l )w/v) Sucrose is also present (preferablyabout 30 g/l).

[0073] Preferably 3-8 grams gellan gum per litre (preferably about 4.5grams) or other gelling agent is also present.

[0074] Preferably it also includes 5 mg/l-50 mg/l (w/v) Abscisic acid(ABA) (preferably about 15 mg/l).

[0075] Preferably the amino acids are present in the following rangesION CONCENTRATION RANGE (mg/l) Arginine  500-2,000 Asparagine1,000-4,000  Glutamine 1,000-10,000 Citrulline 0-50 Ornithine 0-50Lysine 0-50 Alanine 0-50 Proline 0-50

[0076] Preferably arginine is about 700, preferably asparagine is about2,100 and preferably glutamine is about 7,300.

[0077] In yet a further aspect the present invention consists in and/oran initiation and maturation media for embryogenic tissue, said mediumcomprising; Final Rate per Litre (mg) Potassium Nitrate (KNO₃) 1431Magnesium Sulphate (MgSO₄.7H₂O) 400 Sodium Nitrate (NaNO₃) 310 AmmoniumDihydrogen Phosphate (NH₄H₂PO₄) 225 Calcium Chloride (CaCl₂.2H₂O) 25Zinc Sulphate (ZnSO₄.7H₂O) 25 Boric Acid (H₃BO₃) 8.0 Manganese Sulphate(MnSO₄.H₂O) 2.72 Copper Sulphate (CuSO₄.5H₂O) 2.4 Potassium Iodide (KI)1.0 Cobalt Chloride (CoCl₂ 6H₂O) 0.2 Molybdic Acid (Na₂MoO₄.2H₂O) 0.2EDTA - Disodium Salt 40 Iron Sulphate 7H₂O 30 Nicotinic Acid 5.0Thiamine HCl 5.0 Pyridoxine 0.5 Inositol 1000 Arginine 700 Asparagine2100 Glutamine 7300 Citrulline 3.95 Ornithine 3.80 Lysine 2.75 Alanine2.0 Proline 1.75 Abscisic Acid 5 to 50 Sucrose 5,000 to 50,000 GellingAgent (GELRITE ™) 3,000 to 8,000 

[0078] Preferably the sucrose is about 30,000, the gelling agent isGELRITE™ about 4,500, and the abscisic acid is about 15.

[0079] In yet a further aspect the present invention consists in aninitiation embryogenic medium that is also effective as a maturationmedium for initiated and maintained embryogenic tissue resulting fromthe initiation.

[0080] In still a further aspect the present invention consists in amethod of increasing the efficiency of maturation of somatic embryoswhich comprises initiating and maturing the embryogenic tissue on a(preferably substantially common) medium which either

[0081] (a) contains ABA and amino acids,

[0082] (c) is of a composition substantially as follows; Final Rate perLiter (mg) Potassium Nitrate (KNO₃) 1431 Magnesium Sulphate (MgSO₄.7H₂O)400 Sodium Nitrate (NaNO₃) 310 Ammonium Dihydrogen Phosphate (NH₄H₂PO₄)225 Calcium Chloride (CaCl₂.2H₂O) 25 Zinc Sulphate (ZnSO₄.7H₂O) 25 BoricAcid (H₃BO₃) 8.0 Manganese Sulphate (MnSO₄.H₂O) 2.72 Copper Sulphate(CuSO₄.5H₂O) 2.4 Potassium Iodide (KI) 1.0 Cobalt Chloride (CoCl₂.6H₂O)0.2 Molybdic Acid (Na₂MoO₄.2H₂O) 0.2 EDTA - Disodium Salt 40 IronSulphate 7H₂O 30 Nicotinic Acid 5.0 Thiamine HCl 5.0 Pyridoxine 0.5Inositol 1000 Arginine 700 Asparagine 2100 Glutamine 7300 Citrulline3.95 Ornithine 3.80 Lysine 2.75 Alanine 2.0 Proline 1.75 Abscisic Acid 5 to 50 Sucrose 20,000 to 40,000 Gelling Agent (GELRITE ™) 2,500 to8,000

[0083] Preferably the sucrose is about 30,000, the celling agent isGELRITE™ and is about 4,500, and the abscisic acid is about 15.

[0084] Preferably the efficiency is a potentiated increase in theefficiency in that the use of the initiation medium as set out in (c) orcontaining ABA and amino acids or just amino acids increases thepercentage of cell lines produced at initiation of the embryogenictissue. (FIGS. 1-5). The cell lines initiated by the invention that goon to produce mature somatic embryos is also increased.

[0085] The present invention encompasses in the preparation of somaticembryos (particularly of conifers) a merged yet still sequentialinitiation and maturation procedure using a common or substantiallycommon medium.

[0086] The present invention also envisages a procedure of generatingmature somatic embryos which comprises:

[0087] initiation of embryogenic tissue from a source of immatureconifer or other woody species embryos by a procedure of the presentinvention previously defined, and

[0088] maturing at least some of the embryogenic tissue to maturesomatic embryos on the same medium but removing some of the initiatedembryogenic tissue from the medium for bulking up and/or maintenance ona different media or for long term storage by cryo preservation beforeproceeding with additional maturation for producing much larger numbersof mature somatic embryos on the same or similar medium.

[0089] In a further aspect the present invention consists in matureembryos yielded by a method in accordance with the present inventionand/or the use of an initiation and/or maturing media as previously setforth.

[0090] In yet a further aspect the present invention consists in aharvested product of a conifer or other woody species where the seedand/or seedling has resulted from the use of a method of the presentinvention and/or an initiation and/or maturing media as previously setforth.

[0091] In still a further aspect the present invention consists in wood,wood chips or cellulosic fibre derived from such a harvested product.

[0092] Indeed in prior art procedures the stages are as set out below.

[0093] 1 Initiation of embryogenic tissue

[0094] 2. Maintenance of embryogenic tissue (optional cryopreservation)

[0095] 3. Development of embryogenic tissue

[0096] 4. Maturation of somatic embryos

[0097] 5. Starvation and storage of somatic embryos (NZ Patent Appl. No.272210)

[0098] 6. Germination of somatic embryos

[0099] 7. Growth of somatic seedling in greenhouse

[0100] 8. Growth of somatic seedlings in field

[0101] With the adoption of the present invention, initiation andmaturation using the same medium, steps 1 to 4 of such a prior artprocedure merge into a combined initiation and maturation stage, theduration of which is approximately 8-12 weeks.

[0102] 1. Initiation and growth of embryogenic tissue (with optionalcryopreservation) 1 a (optional)Maintenance (with [optional]Cryopreservation)optional Development

[0103] 2. Maturation of somatic embryos

[0104] 3. Starvation and storage of somatic embryos (preferably as inNew Zealand Patent Specification No. 272210)

[0105] 4. Germination of somatic embryos

[0106] 5. Growth of somatic seedling in greenhouse

[0107] 6. Growth of somatic seedlings in field

[0108] Somatic embryos produced by this invention can be used in theperformance of the invention of our New Zealand Patent Specification No.272210.

[0109] The methods and media of the present invention will now bedescribed with particular reference to Pinus radiata as New Zealand'spredominant exotic conifer species.

[0110] For initiation, whole megagametophytes are not used because ofthe uncertainty of whether a seed is fertilised or not. If notfertilised, no useful embryogenic tissue is formed.

[0111] Dissected fertilised embryos at the bullet stage and before thepre-cotyledonary stage, about 500-1000 called embryo head developmentalstage. Other stares of development may be appropriate for other woodyspecies, depending on the size and morphology of fertilised embryos.

[0112] Dissected embryos can be placed directly onto the initiationmedium or onto a nurse culture. Vigorous growth of embryogenic tissueresults.

[0113] Initiation media in at least some preferred forms for Pinusradiaza did not contain traditional/conventional plant growth regulatorssuch as auxins and cytokinins (ie; 2, 4-D, IAA, NAA, IBA and BAP, 2-IP,Zeatin, TDZ etc). The media of the present invention does contain ABAand/or some or all amino acids.

[0114] Typically initiation obtained with this method and of thoseinitiated typically mature. These results have been shown over a rangeof eight genetically different families of Pinus radiata.

[0115] The performance of the present invention will now be describedwith particular reference to the accompanying drawings againstprocedures also for Pinus radiata as disclosed in South African PatentNo. 93/4807.

[0116] In the accompanying drawings

[0117]FIG. 1 is a comparison of initiation efficiencies relative to thetotal seeds extracted between the new method (that of the presentinvention) and the old method (that of South African Patent No.93/4807),

[0118]FIG. 2 is a comparison of initiation efficiencies relative to thenumber of surviving explants again comparing the new method and the oldmethod,

[0119]FIG. 3 is a comparison of contamination methods obtained by eachinitiation method;

[0120]FIG. 4 is a comparison of explants into culture as a measure ofscreening ability; and

[0121]FIG. 5 shows initiation results for various media types.

[0122] Statistical analysis (standard error of the mean) was carried outfor all data in FIGS. 1 to 4.

[0123] Efficiency of initiation for radiata pine was assessed by twomethods, one comparing the efficiency of the two methods relative tototal seeds that were extracted from the cones and the other relative tothe number of explants that survived being placed into culture. The“new” method is that described in the present invention for theinitiation stage only. The “old” method and medium is that described inSouth African Patent No. 93/4807. Initiation for the purposes of FIGS. 1and 2 is classified as embryogenic tissue which has grown well enough tobe classified as an embryogenic cell-line. Comparison of the two methodswas only possible with crosses A96, B96, C96, D96, E96, G96, H96 and J96initiated in late 1995/early 1996.

[0124] The total number of seeds tested for the old method was 16,927for the purpose of FIG. 1 and for the new was 28,414. For the purpose ofFIG. 2 the total number of surviving explants was 7,737 for the old and9,974 for the new methods.

[0125]FIG. 1 shows the difference in initiation efficiency between thetwo methods relative to starting seed counts. This shows that the newmethod has, on average, a significantly high efficiency in formingembryogenic cell lines.

[0126] The overall percentage initiation with the new method was 9.48%compared with 1.61% for the old method. At the individual cross level,the best method was not always the new method, when calculated relativeto starting seed counts, as only crosses C96, E96 and H96 weresignificantly higher in initiation efficiency. This is thought to berelated to other factors inherent to the cone (eg. stage of development,fertilisation efficiency) and to genetic factors.

[0127]FIG. 2, which is a comparison of initiation efficiency relative tothe number of surviving explants, shows a similar trend with the oldmethod still showing a lower initiation efficiency. The overall averagepercentage initiation with the new method was 15.99% compared with 5.99%for the old method.

[0128] Embryogenic tissue of Pinus taeda, Pinus pinaster, Pinustorreyana and Agathis australis has been successfully initiated usingthe same method and media outlined in respect to the present invention.

[0129] The medium of or used by the methods of the present invention isbelieved optimal for Pinus radiata species for both initiation andmaturation but nevertheless has a utility in relation to conifers andwoody species in general, at least in respect of initiation, althoughfor some species maintenance in a healthy state may cause difficultywith a common inination/maturation medium. The Pinus radiata speciesmaintained in the common (preferred) initiation/maturation media, ahealthy state for significant periods and tissue of Pinus pinaster andAgathis australis could be proliferated and maintained in a healthystate for at least four months. Explants for the other species wereselected at a similar stage of development as radiata pine, forinitiation whereby the size and morphology of embryos differed.

[0130] Contamination levels obtained from the two initiation methods(FIG. 3) show highly significant differences depending on the initiationmethod used. This was an unexpected new benefit of the common(substantially common) initiation/maturation media. The old method showsa much higher level of contamination, on average 20 times that of thenew method. The overall average percentage contamination with the oldmethod was 30.32% compared with 1.41% for the new method. The extremelyhigh contamination levels of crosses A96 and C96 were associated withthe use of old initiation media.

[0131] Another way of interpreting the new and old method for initiationis the screening comparisons shown by reference to FIG. 4.

[0132] If an initiation method were to allow explants to be screened forinitiation efficiency, then the use of media would be reduced, resultingin a cost saving. Comparison of the number of seeds initially extractedfrom the cone to the number of explants placed into culture can give ameasure of the difference in the ability of the two methods to allowscreening. The difference between the new and old method (FIG. 4) showsthere is an added ability of the new method to allow screening of.explants. On average the new method allows 25% more screening than theold method. The average percentage of explants that could be placed inculture, if a screening procedure was used, using the new method wouldbe 60.23% whereas with the old method would be 35.54%.

[0133] The addition or deletion of key components to the new initiationmedium in this invention and the effect of these on initiation wasevaluated in order to determine which factors were essential. Fourtreatments were evaluated.

[0134] 1 . Preferred medium containing ABA and amino acids (control).

[0135] 2. Preferred medium containing ammo acids and no ABA.

[0136] 3. Preferred medium containing ABA and no amino acids.

[0137] 4. Preferred medium containing no ABA and no amino acids.

[0138]FIG. 5 demonstrates that the preferred media are those containingboth ABA and amino acids or just amino acids, wherein percentageinitiation values were 40.2% and 43.5% respectively. Media without aminoacids or with no amino acids and no ABA had significantly lowerpercentage initiation at 17.0% and 11.1% respectively. Each media wastested with 4 different crosses.

[0139] The best percentage initiation of embryogenic cell lines fromexplants from the top 10 cones for both “new” and “old” methods (out ofa total of 552 cones) was 42.91% for the new method and 26.29% for theold method. The initiation efficiency was calculated relative to thenumber of surviving explants and was statistically analysed (t−testsignificance<0.003).

[0140] Percentage of cell lines forming mature radiata pine somaticembryos using “old” and “new” methods of initiation No. of MaturePercentage No. of Initiated Embryo-Forming Maturation Cell Line CellLines Efficiency Old* 3642 338 9.3 New** 366 114 31.1

[0141] Data on maturation from “old” method in FIGS. 1-5 not available,data shown for “new” method above is from 4 crosses.

[0142] While in our preferred form of the invention we prefer to use theone medium for both initiation and maturation, the essence of theinvention as claimed in respect of initiation and/or maturation mediaand methods would still be used where a different appropriate nutrientformulation is used for maturation from that used for initiation. Indeedthe present invention envisages the use of an initiation or maturationmedia as claimed or indeed any appropriate initiation or maturationmedia to which, for maturation purposes at least, ABA and/or amino acidshave been added. This is true whether for radiata pine or other coniferor other woody species.

[0143] This procedure of the present invention has an advantage ofensuring greater efficiencies of both initiation and maturation of anyinitiated embryogenic tissue, the clones being passed through the systemto the maturation and beyond to starvation and storage and germination.

1. A method of initiating embryogenic tissue from a source of immatureembryos of a conifer or other woody species, said method comprising:placing explants of the immature embryos on and/or in an initiationmedium or on a nurse callus itself on or in the initiating medium, andallowing sufficient time for initiation to take place, wherein theinitiation medium contains (i) at least one amino acid, or (ii) both ABAand at least one amino acid.
 2. A method of claim 1 wherein the sourceis a conifer.
 3. A method of claim 1 or 2 wherein said explant is notthe whole megagametophyte and is the dissected fertilised embryos at 2to 500 or more—(if a conifer species) celled embryo head stage.
 4. Amethod of claim 3 when dissected at about the bullet or 500-1000-celledembryo head stage.
 5. A method of any one of the preceding claimswherein the initiating medium does not contain traditional/conventionalplant growth regulators such as auxins and cytokinins (eg; 2, 4D, IAA,NAA, IBA, BAP, 2-IP, Zeatin, TDZ, etc) but it does contain ABA and/orone or more amino acids.
 6. A method of any one of the preceding claimswherein ABA (Abscisic Acid) is present.
 7. A method of any one of claims1 to 5 wherein ABA is absent but at least one amino acid is presentbeing one or more of said amino acid(s) Arginine, Asparagine, Glutamine,Citrulline, Ornithine, Lysine, Alanine and Proline.
 8. A method of claim6 or 7 wherein Glutamine is present.
 9. A method of claim 6, 7 or 8wherein at least one of Asparagine and Arginine is present.
 10. A methodof claim 6 or 7 wherein Glutamine, Asparagine and Arginine are present.11. A method of any one of the preceding claims wherein said initiatingmedium includes in addition to said ABA and/or said at least one aminoacid other nutrient sources such as, for example, a source of essentialmacro and micro elements, vitamins, carbohydrates, inositol etc.
 12. Amethod of any one of the preceding claims wherein the initiation mediumincludes inorganic ions in the following concentrations CONCENTRATIONRANGE ION (mmoles/1) NO₃ 4-27 NH₄ 0.5-6.8  Ca  0-0.9 Fe   0-0.15 Na 0-7 Zn   0-0.135 Cu   0-0.05 Mg   0-3.24


13. A method of claim 12 wherein said ion concentrations areCONCENTRATION RANGE ION (mmoles/1) NO₃ about 17.8 NH₄ about 1.96 Caabout 0.17 Fe about 0.10 Na about 3.85 Zn about 0.09 Cu about 9.61 ×10⁻³ Mg about 1.62


14. A method of any one of claims 1 to 11 wherein also present in themedium are the following inorganic ions or the total presence ofinorganic ions is as follows ION CONCENTRATION (mmoles/l) NO₃ 17.80 NH₄1.96 TOTAL N 19.76 P 1.96 K 14.16 Ca 0.17 Mg 1.62 Cl 3.42 × 10⁻¹ Fe 0.10S 1.83 Na 3.85 B 0.13 Mn 1.62 × 10⁻² Zn 0.09 Cu 9.61 × 10⁻³ Mo 8.27 ×10⁻⁴ Co 8.41 × 10⁻⁴ I 6.02 × 10⁻³


15. A method of any one of the preceding claims wherein the mediacontains 5 g/l-50 g/l (w/v) Sucrose.
 16. A method of any one of thepreceding claims wherein the media contains 3-9 grams gellan gum orother gelling agent.
 17. A method of any one of the preceding claimswherein the amino acids are present in the following ranges IONCONCENTRATION RANGE (mg/l) Arginine  500-2,000 Asparagine 1,000-4,000Glutamine 1,000-10,000 Citrulline 0-50 Ornithine 0-50 Lysine 0-50Alanine 0-50 Proline 0-50


18. A method of any one of the preceding claims including allowing atleast partial maturation wherein said initiation media is used as thematuration media.
 19. A method of initiation of embryogenic tissue froma source of immature conifer or other woody species, said methodcomprising: placing explants of the immature fertilised embryos(directly or indirectly eg; nurse callus) on and/or in an initiationmedium, and allowing sufficient time for the initiation to take place,wherein the initiation medium contains (i) amino acids, or (ii) ABA andamino acids.
 20. A method of claim 19 wherein conifers are the source ofthe explants.
 21. A method of claim 19 or 20 wherein the initiationmedium contains ABA.
 22. A method of any one of claims 19 to 21 whereinthe sufficient time is of the order of about 4 weeks.
 23. A method ofany one of claims 19 to 22 wherein the environment is in a steriletissue culture vessel at 15-28° C.
 24. A method of initiation ofembryogenic tissue from a source of immature conifer or other woodyspecies, said method comprising: placing explants of the immaturefertilised embryos (directly or indirectly eg; nurse cells) on and or inan initiation medium, and allowing sufficient time for the initiation totake place, wherein the initiation medium is; Final Rate per Liter (mg)Potasium Nitrate (KNO₃) 1431 Magnesium Sulphate (MgSO₄.7H₂O) 400 SodiumNitrate (NaNO₃) 310 Ammonium Dihydrogen (NH₄H₂PO₄) 225 Phosphate CalciumChloride (CaCl₂.2H₂O) 25 Zinc Sulphate (ZnSO₄.7H₂O) 25 Boric Acid(H₃BO₃) 8.0 Manganese Sulphate (MnSO₄.H₂O) 2.72 Copper Sulphate(CuSO₄.5H₂O) 2.4 Potassium Iodide (KI) 1.0 Cobalt Chloride (CoCl₂.6H₂O)0.2 Molybdic Acid (Na₂MoO₄.2H₂O) 0.2 EDTA-Disodium Salt 40 Iron Sulphate7H₂O 30 Nicotinic Acid 5.0 Thiamine HCl 5.0 Pyridoxine 0.5 Inositol 1000Arginine 700 Asparagine 2100 Glutamine 7300 Citrulline 3.95 Ornithine3.80 Lysine 2.75 Alanine 2.0 Proline 1.75 Abscisic Acid 5 to 50 Sucrose5,000 to 50,000 Gelling Agent (GELRITE ™) 3,000 to 8,000. 


25. A method of producing mature somatic embryos comprising the steps(1) placing explants of the immature embryos on and/or in an initiationmedium or on a nurse callus itself on or in the initiation medium, (2)allowing the initiation to take place, (3) (whether after optionalstorage and maintenance or not) maturing the initiated embryos on anappropriate maturation medium, and wherein (a) the initiating andmaturation medium may be the same or different, and wherein (b) (i) atleast the initiating medium contains ABA and at least one amino acid, or(b) (ii) the initiating medium contains no ABA but includes at least oneamino acid.
 26. A method of claim 25 wherein the source is a conifer.27. A method of claim 26 wherein ABA is present.
 28. A method of claim25, 26 or 27 wherein said at least one amino acid is selected from thegroup Arginine, Asparagine, Glutamine, Citrulline, Ornithine, Lysine,Alanine and Proline.
 29. A method of any one of claims 25 to 28 whereinthe maturation medium contains ABA and at least one amino acid. 30.Embryos matured by a method or using a method of any one of thepreceding claims.
 31. An initiation and/or maturation media forembryogenic tissue, said medium comprising in addition to a presence ofABA and at least one amino acid inorganic ions in the followingconcentrations ION CONCENTRATION RANGE (mmoles/1) NO₃  4-27 NH₄ 0.5-6.8Ca 0.01-0.9  Fe 0.025-0.15  Na 0.5-7   Zn 0.023-0.135 Cu 6 × 10⁻⁴-5 ×10⁻² Mg 0.405-3.24 


32. A media of claim 31 wherein said ion concentrations are IONCONCENTRATION RANGE (mmoles/l) NO₃ about 17.8 NH₄ about 1.96 Ca about0.17 Fe about 0.10 Na about 3.85 Zn about 0.09 Cu about 9.61 × 10⁻³ Mgabout 1.62


33. A media of claim 31 or 32 wherein said ion concentrations are asfollows Final Rate per Liter (mg) Potassium Nitrate (KNO₃) 1431Magnesium Sulphate (MgSO₄.7H₂O) 400 Sodium Nitrate (NaNO₃) 310 AmmoniumDihydrogen Phosphate (NH₄H₂PO₄) 225 Calcium Chloride (CaCl₂.2H₂O) 25Zinc Sulphate (ZnSO₄.7H₂O) 25 Boric Acid (H₃BO₃) 8.0 Manganese Sulphate(MnSO₄.H₂O) 2.72 Copper Sulphate (CuSO₄.5H₂O) 2.4 Potassium Iodide (KI)1.0 Cobalt Chloride (CoCl₂.6H₂O) 0.2 Molybdic Acid (Na₂MoO₄.2H₂O) 0.2EDTA - Disodium Salt 40 Iron Sulphate 7H₂O 30 Nicotinic Acid 5.0Thiamine HCl 5.0 Pyridoxine 0.5 Inositol 1000 Arginine 700 Asparagine2100 Glutamine 7300 Citrulline 3.95 Ornithine 3.80 Lysine 2.75 Alanine2.0 Proline 1.75 Abscisic Acid 5 to 50 Sucrose 5,000 to 50,000 GellingAgent (GELRITE ™) 3,000 to 8,000 


34. A media of any one of claims 31 ro 33 wherein 5 g/l-50 g/l )w/v)Sucrose is also present.
 35. A media of any one of claims 31 to 34wherein 3-9 grams gellan gum per litre is present.
 36. A media of anyone of the preceding claims wherein amino acids are present in thefollowing ranges ION CONCENTRATION RANGE (mg/l) Arginine  500-2,000Asparagine 1,000-4,000  Glutamine 1,000-10,000 Citrulline 0-50 Ornithine0-50 Lysine 0-50 Alanine 0-50 Proline 0-50


37. An initiation and maturation media for embryogenic tissue, saidmedium comprising; Final Rate per Liter (mg) Potassium Nitrate (KNO₃)1431 Magnesium Sulphate (MgSO₄.7H₂O) 400 Sodium Nitrate (NaNO₃) 310Ammonium Dihydrogen Phosphate (NH₄H₂PO₄) 225 Calcium Chloride(CaCl₂.2H₂O) 25 Zinc Sulphate (ZnSO₄.7H₂O) 25 Boric Acid (H₃BO₃) 8.0Manganese Sulphate (MnSO₄.H₂O) 2.72 Copper Sulphate (CuSO₄.5H₂O) 2.4Potassium Iodide (KI) 1.0 Cobalt Chloride (CoCl₂.6H₂O) 0.2 Molybdic Acid(Na₂MoO₄.2H₂O) 0.2 EDTA - Disodium Salt 40 Iron Sulphate 7H₂O 30Nicotinic Acid 5.0 Thiamine HCl 5.0 Pyridoxine 0.5 Inositol 1000Arginine 700 Asparagine 2100 Glutamine 7300 Citrulline 3.95 Ornithine3.80 Lysine 2.75 Alanine 2.0 Proline 1.75 Abscisic Acid 5 to 50 Sucrose5,000 to 50,000 Gelling Agent (GELRITE ™) 3,000 to 8,000.


38. An initiation embryogenic medium that is also effective as amaturation medium for initiated and maintained embryogenic tissueresulting from the initiation, said medium being as claimed in any oneof claims 31 to
 37. 39. A method of increasing the efficiency ofmaturation of somatic embryos which comprises initiating and maturingthe embryogenic tissue on a substantially common medium which either (a)contains ABA and amino acids, (b) is of a composition substantially asfollows; Final Rate per Liter (mg) Potassium Nitrate (KNO₃) 1431Magnesium Sulphate (MgSO₄.7H₂O) 400 Sodium Nitrate (NaNO₃) 310 AmmoniumDihydrogen Phosphate (NH₄H₂PO₄) 225 Calcium Chloride (CaCl₂.2H₂O) 25Zinc Sulphate (ZnSO₄.7H₂O) 25 Boric Acid (H₃BO₃) 8.0 Manganese Sulphate(MnSO₄.H₂O) 2.72 Copper Sulphate (CuSO₄.5H₂O) 2.4 Potassium Iodide (KI)1.0 Cobalt Chloride (CoCl₂.6H₂O) 0.2 Molybdic Acid (Na₂MoO₄.2H₂O) 0.2EDTA - Disodium Salt 40 Iron Sulphate 7H₂O 30 Nicotinic Acid 5.0Thiamine HCl 5.0 Pyridoxine 0.5 Inositol 1000 Arginine 700 Asparagine2100 Glutamine 7300 Citrulline 3.95 Ornithine 3.80 Lysine 2.75 Alanine2.0 Proline 1.75 Abscisic Acid 5 to 50 Sucrose 5,000 to 50,000 GellingAgent (GELRITE ™) 3,000 to 8,000


40. A method of claim 40 wherein (a) or (c) is used.
 41. A method ofclaim 40 or 41 wherein (c) is used and the sucrose is about 30.000 andthe gelling agent is GELRITE™ about 4,500.
 42. A method of any one ofclaims 39 to 41 wherein the efficiency is a potentiated increase in thepercentage of clones producing mature embryos from embryogenic tissuethe embryogenic tissue having been initiated as in claim 1-24.
 43. Amethod of any one of claims 39 to 42 wherein the efficiency is as aresult of reduced contamination owing to the use of the same media forinitiation and maturation.